In vivo delivery of engineered tRNAs for suppression of nonsense mutations

翻译后摘要：无义突变改变氨基酸密码子提前终止密码子（PTC），导致 在一个有缺陷的截短蛋白质和严重的疾病形式。无义突变占10个以上 占所有遗传性疾病的90%，占近1，000种人类遗传性疾病，包括囊性纤维化 （CF）。事实上，10%的CF患者有无义突变（1型突变），导致过早的 囊性纤维化跨膜传导调节因子（CFTR）蛋白的截短和 无义介导的衰变（NMD）。最常见的无义突变包括G542 X， R553 X、R1162 X和W1282 X占所有CF无义突变的10%，导致CFTR丢失 最严重的CF表型。CFTR蛋白是一种氯离子通道，其在CF中的缺失 改变肺衬里液的正常稳态，导致高度粘稠的粘液，使细菌感染 最终导致致命的感染预测肺中10%至15%的突变CFTR的校正 是有效治疗这种疾病的门槛。而氨基糖苷类和非氨基糖苷类小- 已经开发了分子，并允许通读PTC、耳毒性和肾毒性， 氨基糖苷类药物的使用和其他药物的不良活性限制了它们的临床应用。我们 我最近开发了一个反密码子编辑（ACE）-tRNA库，该库识别并促进通读 所有“帧内”PTC。每种ACE-tRNA都有一个识别PTC的单位点突变，但ACE- tRNA上带有一个氨基酸以通读PTC。我们利用了小tRNA 表达盒（~72 bp），并产生了几个紧凑的DNA载体，我们称之为微型载体。带有图书馆 在>500个ACE-tRNA中，我们可以将任何所需的氨基酸插入任何PTC中。我们已经证明，这种方法 在体外和体内都能高效地拯救含有PTC的发光报告基因， 以及CRISPRed 16 HBE 140-细胞中基因组背景内的G542 X-、R1162 X-和W1282 X-CFTR，导致 NMD和CFTR蛋白功能的校正。我们的目标是测试有效性，行动的持久性， 在人气道上皮细胞中作为PTC通读和校正的小载体递送的ACE-tRNA的安全性 PTC细胞培养模型和在猪和CFTR PTC突变小鼠的肺中的体内研究。为了将这些ACE-tRNA 小鼠和猪的肺，我们将使用经胸电穿孔，这已被证明是安全，简单， 并且高效地将基因传递到肺部，包括气道。总体目标是确定 ACE-tRNA用于治疗无义相关疾病的治疗前景。

Abstract: Nonsense mutations change an amino acid codon to a premature termination codon (PTC), resulting in a defective truncated protein and severe forms of disease. Nonsense mutations account for greater than ten percent of all genetic diseases, accounting for nearly 1,000 genetic human disorders, including cystic fibrosis (CF). Indeed, 10% of people with CF have nonsense mutations (Type 1 mutations) that lead to premature truncation of the cystic fibrosis transmembrane conductance regulator (CFTR) protein and significant loss of transcripts due to nonsense mediated decay (NMD). The most common nonsense mutations include G542X, R553X, R1162X and W1282X, which account for 10% of all CF nonsense mutations, result in loss of CFTR function and the most severe CF phenotypes. The CFTR protein is a chloride channel whose absence in CF alters normal homeostasis of lung lining fluid, resulting in highly viscous mucus that allows bacterial infection and ultimately lethal infections. Correction of between 10 and 15% of the mutant CFTR in the lung is predicted to be the threshold for effective treatment of the disease. While aminoglycosides and non-aminoglycoside small- molecules have been developed and allow readthrough of PTCs, ototoxicity and nephrotoxicity with extended use in the case of aminoglycosides and poor activity for the other drugs has restricted their use clinically. We have recently developed a library of Anti-Codon Edited (ACE)-tRNAs that recognize and promote read-through of all `in-frame' PTCs. Each of the ACE-tRNAs has a single site mutation that recognizes the PTC but the ACE- tRNA is charged with an amino acid to readthrough the PTC. We have taken advantage of the small tRNA expression cassette (~72 bp), and generated several compact DNA vectors, we call minivectors. With a library of >500 ACE-tRNAs, we can insert any desired amino acid into any PTC. We have shown that this approach works with high efficiency both in vitro and in vivo in rescuing PTC-containing luminescent reporter genes, as well as G542X-, R1162X- and W1282X-CFTR within the genomic context in CRISPRed 16HBE14o- cells leading to correction of both NMD and CFTR protein function. Our goal is to test the efficacy, persistence of action and safety of ACE-tRNAs delivered as minivectors for PTC readthrough and correction in human airway epithelial PTC cell culture models and in vivo in lung of pig and CFTR PTC mutant mice. To deliver these ACE-tRNA to the lungs of mice and pigs, we will use transthoracic electroporation which has been shown to be safe, simple, and highly efficient at gene delivery to the lung, including the airways. The overarching goal is to determine the therapeutic promise of ACE-tRNAs for treatment of nonsense associated diseases.